Functional Analysis Of Zinc Finger Gene GhRCHY1 Related To Droght Tolerance In Cotton

Cotton is an important fiber crop in the world.China is among the major cotton producing,consuming and textile exporting countries.With the modularization and regionalization development of agricultural industrial,Xinjiang is becoming the major cotton-producing province in China due to favorable environmental and climatic conditions,therefore,there is a higher requirement for the breeding goal to develop drought-tolerant cotton varieties.Zinc finger proteins（ZFNs）are type proteins that can chelate with zinc ions and form a"finger"structure.ZFNs are primarily composed of cysteine and histidine combined with zinc ions in a specific number,order and configuration.ZFNs can perform various biological functions based on multivalent activity of zinc ions.Previous study in our group inicateded that the expresson of GhRCHY1 with a 945bp long ORF encoding 315 amino acids,was significantly induced by drought and created overexpressed GhRCHY1 with high generation and pure cotton lines.In the current study,R-1,R-2,R-3 lines were used as research materials and the wild-type W0 as the control.The role of GhRCHY1 in drought tolerance was elucidated based on morphological and physiological characterization,expression analysis and transcriptome analysis.The main results are as follows:1.Expression analysis of GhRCHY1:tissue-expression analysis was conducted by using the c DNA of the root,stem,leaf,ovule and fiber tissues.Quantitative PCR results showed that expression of GhRCHY1 varied among various tissues.In roots of the seedlings grown under the treatments of 100 u M ABA,15%PEG6000 and 200 m M Na Cl,the expression of GhRCHY1 at 2 h,4 h,6 h,8 h and 12 h was significantly increased as compared to 0h,indicating that the expression of GhRCHY1 was significantly induced by abiotic stresses.2.GhRCHY1 subcellular localization and self-activation activity analysis:the full length open reading frame of GhRCHY1 without stop codon fused with GFP subcellular localization expression vector was constructed and injected into tobacco through Agrobacterium-mediated transformation;and the results were observed by using laser confocal microscope.The results showed that 35S:GhRCHY1-GFP is distributed in the nucleus,cell membrane and cytoplasm.The results of Yeast self-activation activity experiment showed that the full length of GhRCHY1 has strong activation activity.Analysis of the protein sequence of GhRCHY1 showed that there is a RING-type domain at its N-terminus,and designing different fragments based on the nucleic acid position corresponding to this domain.Yeast self-activation activity verification found that the segment containing the RING-type domain is the key activation activity segment.3.Positive identification of GhRCHY1 transgenic cotton material:the 3 lines of genetically modified materials（R-1,R-2,R-3）,were evaluated at DNA level by using promoter/gene specific primers andthe primers of the gene NPTII（kanamycin as a selection marker）and the reporter gene GUS were applied to examine the stability of genetic integration of high-generation materials.The number of inserted copies of the foreign fragments of the overexpression strains was validated through digital drop PCR,showing multiple copies in R-1 and R-2,and singly copy in R-3.The overexpression of GhRCHY1in leaves and roots at the m RNA level was further verified by q RT-PCR.4.Morphological and physiological impact of overexpression of GhRCHY1:the overexpression material was subjected to natural drought while wild-type W0 material was used as a control.Overexpression material exhibited higher drought tolerance by maintaining higher relative leaf water contents and a slower water loss rate as compared to W0.Photosynthetic parameters,such as net photosynthetic rate,transpiration rate,stomatal conductance,maximum photon yield Fv/Fm and actual photon yield were significantly higher in overexpressed line than the control W0,while the intercellular CO₂concentration was significantly lower than the W0.Physiological and biochemical indicators,such as SOD activity was significantly higher than the control,while the content of MDA and H₂O₂were significantly lower than the control in overexpressed material.After silencing GhRCHY1 through VIGS,the tolerance to drought stress was significantly reduced,and the photosynthetic index,physiological and biochemical index parameters were opposite to the overexpression material.These results indicated that GhRCHY1 plays a positive regulatory role in the response to natural drought in cotton.5.The effect of GhRCHY1 on root morphology at seedling stage in cotton:The phenotypic differences were observed in the root morphology between overexpression material and the wild-type W0.The number of root tips of the overexpression material was significantly more than that of W0 under well-watered conditions,but after drought treatment,the average root diameter,root length and root volume were also increased significantly in overexpression lines as compared to W0.After silencing GhRCHY1,the average diameter,length and volume of the roots were reduced to varying degrees,indicating that GhRCHY1 may directly affected the growth and development of root indices in cotton.6.Mechanism of GhRCHY1 in regulation of drought tolerance:transcriptome analysis was performed on the root tissues of the overexpressed transgenic material R-3 and the control W0 under drought treatment.1770 differentially expressed genes were identified between R-3 and W0,out of which,1245 genes are up-regulated in R-3,and 525 genes are down-regulated in R-3.GO enrichment indicated that the 1245 genes up-regulated were mainly involved in cell wall organization and biosynthesis,polysaccharide metabolism and carbohydrate metabolism while the down-regulated 525 genes were mainly enriched in hydrogen peroxide metabolism,reactive oxygen metabolism and redox processes,etc.metabolic pathway,which indicated that the overexpression of GhRCHY1 affects the biosynthesis and redox process of the cell wall of root tissue.Comparing the transcriptome of R-3 under drought treatment and W0 drought treatment,it was found that there were3646 up-regulated differentially expressed genes,and then combined with the 1770up-regulated differentially expressed genes of R-3 and W0 under water treatment conditions,588 up-regulated differentially expressed genes were found.Expressed genes are defined as genes that overexpressing GhRCHY1 and are up-regulated in response to drought.GO enrichment analysis of these 588 genes reveals that they mainly involve pathways such as hormone metabolism,microtubule formation,and hormone response detection.Combined with the root morphological data,the genes involved in the hormone metabolism pathway were analyzed,and it was found that they were mainly related to cytokinin metabolism,and cytokinins mainly affected the formation of lateral roots.Quantitative verification and hormone content determination were carried out.It was found that the expression of genes related to cell classification in the overexpression material was significantly higher than that of the control W0,and the zeatin content was significantly lower than that of the control W0.These results indicate that GhRCHY1 may improve the tolerance of plants to drought stress by changing the components of plant root tissues,cell walls and regulating the levels of cytokine to change root morphology.